Inhibition of interleukin-6 synthesis in an animal model of septic shock by anti-C5a monoclonal antibodies

The complement activation fragment C5a was recently shown to induce interleukin (IL)-6 synthesis by peripheral blood mononuclear cells. To understand better the role of C5a in cytokine regulation in vivo, we investigated the effects of complement depletion by cobra venom factor (CVF) or of anti-C5a monoclonal antibodies (mAb) on IL-6 generation in an animal model of septic shock. Complement-depleted pigs which were subsequently challenged with Escherichia coli generated significantly (p < 0.05) less IL-6 during the 6-h observation period than complement-sufficient controls. To address specifically the role of C5a in IL-6 regulation, we produced a C5a(57–74) peptide-specific mAb (T13/9) which neutralizes the bioactivity of porcine C5a. The mAb T13/9 does not crossreact with the precursor protein C5. The pretreatment of pigs with anti-C5a mAb T13/9 prior to the induction of sepsis resulted in a decrease of over 75 % in serum IL-6 bioactivity compared to control animals (p < 0.0001). These results indicate a role for C5a in the modulation of IL-6 synthesis in Gram-negative bacteremia.     

Applications of a rat multiple tissue gene expression data set

With the sequencing and assembly of the rat genome comes the difficult task of assigning functions to genes. Tissue localization of gene expression gives some information about the potential role of a gene in physiology. Various examples of the utility of multiple tissue gene expression data sets are illustrated here. First, we highlight their use in finding genes that might play an important role in a particular tissue on the basis of exclusive expression in that tissue or coexpression with a gene or genes with known function. Second, we show how this data might be used to explain known phenotypic differences between strains. Third, we show how expression patterns of genes in a genomic interval might identify candidate genes in quantitative trait loci (QTL) mapping studies. Lastly, we show how multiple tissue and species data can help researchers prioritize follow up studies to microarray experiments. All of these applications of multiple tissue gene expression data sets will play a role in functionally annotating the rat genome.     

Corneal crystals in nephropathic cystinosis: natural history and treatment with cysteamine eyedrops

Although renal disease is the most prominent feature of the lysosomal storage disease cystinosis, corneal cystine crystal formation remains a major complication, leading to photophobia, corneal erosions, and keratopathies. Moreover, the extent of corneal crystal accumulation reflects the course and severity of the disease itself, and the cornea is accessible to direct examination. Therefore, we employed a scoring system, based on a library of slit-lamp photographs of corneas with increasing crystal densities (0.00-3.00), to assess the degree of crystal accumulation in 170 patients with nephropathic cystinosis examined at the National Institutes of Health between 1976 and 2000. None of the patients had received topical cystine-depleting therapy at the time of the evaluation. In this natural history study, infants in the first year of life had absent or minimal corneal crystals, i.e., a corneal cystine crystal score (CCCS) of 0 or 0.25. However, the CCCS increased linearly with age, such that every patient had visible crystals by 16 months of age, and plateaued at approximately 3.00 by early adolescence. Longitudinal studies in representative patients support the cross-sectional results. Individuals homozygous for the common 57-kb deletion involving the cystinosis gene (CTNS) displayed the same course of corneal crystal accumulation as did individuals not bearing the large deletion. Patients with ocular or nonnephropathic cystinosis had CCCSs that were, in general, half those expected for patients with nephropathic cystinosis of the same age. Administration of 0.55% cysteamine eyedrops, given 6 to 12 times per day, dissolved corneal cystine crystals in 10 representative patients with nephropathic cystinosis aged 1 to 32 years within 8 to 41 months.     

Regional distribution of fiber types in developing baboon diaphragm muscles

Fiber type distribution and mean fiber area were determined for seven sites in diaphragm muscles of premature (140 days gestation), full-term (180 days gestation), and adult baboons. Within a group, data did not differ significantly amongst the seven sites. The diaphragm of premature animals had a large proportion [56(+/- 2)%] of type IIc fibers, smaller proportions of type I, IIo, and IIh fibers [16(+/- 2), 21(+/- 1), and 7(+/- 2)%, respectively], and no type IIg fibers. Full-term animals had fewer type IIc [2(+/- 1)%] fibers, greater proportions of type I [46(+/- 2)%], IIh [23(+/- 1)%], and IIg [11(+/- 1)%] fibers, and a similar proportion of type IIo fibers [17(+/- 1)%]. Diaphragm from adult baboons had similar proportions of type IIh, IIg, and IIc fibers in females [39(+/- 4), 20(+/- 2), 1(+/- 1), 41(+/- 5), and 1(+/- 1)%] and males [48(+/- 2), 16 (+/- 1), 0(+/- 0), 36(+/- 2), and 3(+/- 2)%]. Fiber area for premature [143(+/- 9), 210(+/- 15), 231(+/- 15), and 156(+/- 16) microns2 for type I, IIo, IIh, and IIc fibers], newborn [317(+/- 32), 374(+/- 36), 468(+/- 42), 498(+/- 43), and 322(+/- 37) microns2 for type I, IIo, IIh, IIg, and IIc fibers], and for type I, IIo, IIg, and IIc fibers from adult female [1,759(+/- 130), 2,365(+/- 284), 5,026(+/- 742), and 1,843(+/- 111) microns2] and adult male [2,513(+/- 221), 3,987(+/- 267), 6,102(+/- 376), and 2,833(+/- 151) microns2] baboons indicated growth which correlated with body weight. Our results also show that metabolic and contractile enzymes develop normally, but growth of respiratory muscle fibers is arrested, during 10 days following premature birth.     

A Randomized Clinical Trial of Topical Cysteamine Disulfide (Cystamine) versus Free Thiol (Cysteamine) in the Treatment of Corneal Cystine Crystals in Cystinosis

In nephropathic cystinosis, corneal cystine crystals cause severe photophobia and corneal erosions. Topical cysteamine dissolves these crystals, but cannot be marketed because it rapidly oxidizes to the disulfide form, cystamine, at room temperature. Since cystamine itself could be used commercially, we compared the efficacy of cystamine and cysteamine with respect to cystine crystal dissolution in a randomized, double-masked clinical trial. One eye each of 14 patients with cystinosis was randomized to either cystamine or cysteamine, 0.5%, with 0.01% benzalkonium chloride; the companion eye was treated with the alternate preparation. Corneal crystals were photographed and a density score was assigned to each slide based on 13 standard slides. After 8–20 months, 6 patients showed significant reduction of the corneal crystal score in only one eye. In each case, the improved eye was the cysteamine-treated eye. Theoretically, cysteamine should dissolve both intracellular and extracellular crystals, whereas cystamine should dissolve only intracellular crystals because it must first be reduced to the free thiol by the cytoplasmic-reducing environment. Hence, the lack of efficacy of the disulfide cystamine suggests that some corneal cystine crystals in cystinosis patients are extracellular, and that another form of stable, topical cysteamine must be developed for cystinosis patients.     

Spectral karyotyping combined with locus-specific FISH simultaneously defines genes and chromosomes involved in chromosomal translocations

Genes that play roles in malignant transformation have often been found proximate to cancer-associated chromosomal breakpoints. Identifying genes that flank chromosomal reconfigurations is thus essential for cancer cytogenetics. To simplify and expedite this identification, we have developed a novel approach, based on simultaneous spectral karyotyping and fluorescence in situ hybridization (FISH) which, in a single step, can identify gross chromosomal aberrations as well as detect the involvement of specific loci in these rearrangements. Signals for specifically queried genes (FISH probe) were easily detectable in metaphase cells, together with the signals from painted chromosomes (spectral karyotyping probes). The concentration and size of the FISH probes could cover a wide range and still be used successfully. Some of the nucleotide-bound dyes used for the labeling, as Cy3, Spectrum Orange, Alexa 594, Texas Red, and Rhodamine 110, were particularly efficient. More than one gene can be queried in the same metaphase, because multiple FISH probes could be hybridized simultaneously. To demonstrate this technique, we applied it to the myeloma cell line Karpas 620, which has numerous chromosomal rearrangements. The approach that we present here will be particularly useful for the analysis of complex karyotypes and for testing hypotheses arising from cancer gene expression studies. Published 2000 Wiley-Liss, Inc.     

The Bioperl toolkit: Perl modules for the life sciences

The Bioperl project is an international open-source collaboration of biologists, bioinformaticians, and computer scientists that has evolved over the past 7 yr into the most comprehensive library of Perl modules available for managing and manipulating life-science information. Bioperl provides an easy-to-use, stable, and consistent programming interface for bioinformatics application programmers. The Bioperl modules have been successfully and repeatedly used to reduce otherwise complex tasks to only a few lines of code. The Bioperl object model has been proven to be flexible enough to support enterprise-level applications such as EnsEMBL, while maintaining an easy learning curve for novice Perl programmers. Bioperl is capable of executing analyses and processing results from programs such as BLAST, ClustalW, or the EMBOSS suite. Interoperation with modules written in Python and Java is supported through the evolving BioCORBA bridge. Bioperl provides access to data stores such as GenBank and SwissProt via a flexible series of sequence input/output modules, and to the emerging common sequence data storage format of the Open Bioinformatics Database Access project. This study describes the overall architecture of the toolkit, the problem domains that it addresses, and gives specific examples of how the toolkit can be used to solve common life-sciences problems. We conclude with a discussion of how the open-source nature of the project has contributed to the development effort.     

Performance of the syva direct fluorescent antibody assay for Chlamydia in a low-prevalence population

Chlamydia trachomatis is the most common reportable sexually transmitted disease (STD) in the United States. In the 1980s, rapid diagnostic tests for chlamydia began to replace more cumbersome tissue culture methods. Current data on rapid antigen detection assays demonstrate acceptable sensitivity, specificity, and predictive values in populations with a high prevalence of chlamydia. Few studies report the performance of these assays in a low-prevalence obstetric and gynecologic (Ob/Gyn) population, This study compares the most commonly used direct fluorescent antibody (DFA) assay (Syva Microtrak) with tissue culture (TC) in a low-prevalence population. Endocervical specimens (775) were tested from women at risk for chlamydia infection, and the prevalence was found to be 7.7%. The DFA assay demonstrated a sensitivity of 80% and a specificity of 97% compared with TC. The positive and negative predictive values were 72% and 98%, respectively. The results of this study indicate that the Syva DFA assay lacks the sensitivity and positive predictive value for routine use in Ob/Gyn populations with a lowprevalence of C. trachomatis.     

Evaluating sex chromosome content of sorted human sperm samples with use of dual-color fluorescence in situ hybridization

OBJECTIVE: Although most methods for selecting the sex of offspring by sorting spermatozoa are ineffective at shifting the ratio of Y- to X-containing cells, some commercial sources continue to offer such services. Our objective was to evaluate commercially “sorted” samples with use of dual-color fluorescence in situ hybridization and to identify variations in assessment by comparing motile and total sperm populations, donors, observers, and fluorescence in situ hybridization probes.STUDY DESIGN: Cryopreserved sperm from seven anonymous donors were processed as for insemination. Sperm cells from each total sample or motile subfraction were prepared for fluorescence in situ hybridization by incubation with disulfide-reducing agents to expand sperm nuclei. Two sets of X and Y chromosome–specific, fluorophore-labeled deoxyribonucleic acid probes were used. At least 400 nuclei from each preparation were classified independently by three blinded observers. Hybridization efficiency, aneuploidy, and sex chromosome content were evaluated in subsets of five unsorted, five female-oriented, and five male-oriented samples. Total and motile subfractions were compared with eight samples. Fluorescence in situ hybridization probes were compared in five paired unsorted samples.RESULTS: No differences were detected between washed samples and paired motile subfractions. No differences in hybridization and aneuploidy were detected between groups of sorted samples. The Y/X ratio was significantly different between the sorted groups. However, male-oriented samples had a lower Y/X ratio than female-oriented samples did. Observer and probe choice accounted for small but significant variations that did not alter conclusions about the Y/X ratio for sorted samples.CONCLUSION: In a series of 10 sorted samples from one commercial source, dual-color fluorescence in situ hybridization demonstrated a small but significant shift in the sex chromosome ratios among samples. However, this shift was opposite to that expected by the orientation of the sorted samples. (Am J Obstet Gynecol 1997;176:1172-80.)